Simultaneous monitoring of faecal indicators and harmful algae using an in-situ autonomous sensor.

UNLABELLED: Faecal indicator bacteria (FIB) and harmful algal blooms (HABs) threaten the health and the economy of coastal communities worldwide. Emerging automated sampling technologies combined with molecular analytical techniques could enable rapid detection of micro-organisms in-situ, thereby improving resource management and public health decision-making. We evaluated this concept using a robotic device, the Environmental Sample Processor (ESP). The ESP automates in-situ sample collection, nucleic acid extraction and molecular analyses. Here, the ESP measured and reported concentrations of FIB (Enterococcus spp.), a microbial source-tracking marker (human-specific Bacteriodales) and a HAB species (Psuedo-nitzschia spp.) over a 45-day deployment on the Santa Cruz Municipal Wharf (Santa Cruz, CA, USA). Both FIB and HABs were enumerated from single in-situ collected water samples. The in-situ qPCR efficiencies ranged from 86% to 105%, while the limit of quantifications during the deployment was 10 copies reaction(-1) . No differences were observed in the concentrations of enterococci, the human-specific marker in Bacteroidales spp., and P. australis between in-situ collected sample and traditional hand sampling methods (P > 0·05). Analytical results were Internet-accessible within hours of sample collection, demonstrating the feasibility of same-day public notification of current water quality conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first report of in-situ qPCR enumeration of both faecal indicators and harmful algal species in coastal marine waters. We utilize a robotic device for in-situ quantification of enterococci, the human-specific marker in Bacteriodales and Pseudo-nitzschia spp. from the same water samples collected and processed in-situ. The results demonstrate that rapid, in-situ monitoring can be utilized to identify and quantify multiple health-relevant micro-organisms important in water quality monitoring and that this monitoring can be used to inform same-day notifications.

Sequestration of soil nitrogen as tannin-protein complexes may improve the competitive ability of sheep laurel (Kalmia angustifolia) relative to black spruce (Picea mariana).

The role of litter tannins in controlling soil nitrogen (N) cycling may explain the competitive ability of Kalmia relative to black spruce (Picea mariana), although this has not been demonstrated experimentally. Here, the protein-precipitation capacities of purified tannins and leaf extracts from Kalmia and black spruce were compared. The resistance to degradation of tannin-protein precipitates from both species were compared by monitoring carbon (C) and N dynamics in humus amended with protein, purified tannins or protein-tannin precipitates. The purity of the precipitates was verified using solid-state (13)C nuclear magnetic resonance (NMR) spectra. The ability of mycorrhizal fungi associated with both species to grow on media amended with tannin-protein complexes as the principal N source was also compared. The protein precipitation capacity of Kalmia tannins was superior to those of black spruce. Humus amended with protein increased both mineral and microbial N, whereas humus amended with tannin-protein precipitates increased dissolved organic N. Mycorrhizal fungi associated with Kalmia showed better growth than those associated with black spruce when N was provided as tannin-protein precipitates. These data suggest that Kalmia litter increases the amount of soil N sequestered as tannin-protein complexes, which may improve the competitive ability of Kalmia relative to black spruce by favouring N uptake by mycorrhizas associated with the former.

Leaching of nitrogen and phenolics from wood waste and co-composts used for road rehabilitation.

Rehabilitation and reforestation of disused forest roads and landings can be facilitated by the incorporation of organic matter. The British Columbia forest industry creates residual woody materials, but they are nutrient poor and may leach phenolic compounds. We assessed the potential for wood wastes (chipped cedar wood waste, sort-yard waste, hogfuel) and co-composts with shellfish waste or municipal biosolids to provide inorganic N and release phenolics and condensed tannins, compared with natural forest floor and mineral soil. Initial concentrations of tannins and phenolics were low, and 13C cross-polarization and magic-angle spinning nuclear magnetic resonance spectroscopy showed that composts were still dominated by wood. During a 426-d laboratory leaching experiment, release of phenolics from woody amendments (other than cedar wood) was lower than from native forest floor. The pH levels of woody amendments and their leachates were also within the range of native forest floor and soil (except cedar wood, which was the most acidic material). Co-composts had higher total N and available P, greatly reduced tannins and phenolics, and negligible leaching of polyphenols. Uncomposted materials released very little N during the incubation. Hogfuel-biosolids compost released a large amount of nitrate, but only during the first 100 d. Shrimp-wood compost released moderate amounts of ammonium and nitrate throughout the incubation, had high available P and low tannin content, and released less polyphenols than did native forest floors. Our results indicate that appropriate use of these amendments does not pose an environmental risk with regard to the parameters measured in this study.

Identification of herpes simplex virus DNA sequences which encode a trans-acting polypeptide responsible for stimulation of immediate early transcription.

Herpes simplex virus (HSV) immediate early (IE) transcription is known to be stimulated by a structural component of the virion which interacts, either directly or indirectly, with specific regulatory sequences located far upstream from IE messenger RNA 5'-termini. The aim of the work described in this paper is the mapping and identification of the virion component. Cloned HSV DNA fragments derived from various parts of the genome were cotransfected into BHK cells together with chimaeric plasmids which contained the thymidine kinase gene under IE control. Stimulation of thymidine kinase synthesis was elicited by cloned EcoRIi (0.63 to 0.72 map units), BamHIf (0.64 to 0.69) or EcoRIb (0.72 to 0.87). Cloned BamHIf had the same specificity as the virion component, since it stimulated thymidine kinase expression only from chimaeric plasmids which contained functional IE-specific regulatory sequences. The effect of EcoRIb was not confined to plasmids with IE-specific regulatory regions, suggesting a more general stimulatory role for one or more of the polypeptides encoded by this fragment. A subclone containing a 2.7 X 10(3) base-pair fragment of BamHIf (pMC1) was active in the cotransfection assay, and the effect was abolished by an eight base-pair insertion into the middle of this fragment. The only polypeptide known to map entirely within the HSV genome region defined by pMC1 was identified as the major tegument species Vmw65. The results therefore suggest that Vmw65 is the virion component which trans-activates HSV IE transcription.

Towards an understanding of the molecular basis of herpes simplex virus latency.

The survival strategy of herpes simplex virus centres on the establishment of latency in sensory neurons innervating the site of primary infection followed by periodic reactivation to facilitate transmission. This is a highly evolved and efficient survival mechanism, which despite being the subject of intense research, has proven remarkably difficult to dissect at a molecular level. This review will focus on data, emerging from both in vitro and in vivo model systems, which provide a framework for a mechanistic understanding of latency and the existence and possible significance of non-uniform latent states.

Use of recombinant vectors derived from herpes simplex virus 1 mutant tsK for short-term expression of transgenes encoding cytoplasmic and membrane anchored proteins in postmitotic polarized cortical neurons and glial cells in vitro.

We constructed three recombinant vectors derived from the herpes simplex virus type 1 mutant tsK, each of which contained a different transgene under the control of the herpes simplex virus type 1 immediate early 3 promoter inserted into the thymidine kinase locus: the prokaryotic enzymes beta-galactosidase and chloramphenicol acetyl transferase, and a fusion gene consisting of human tissue inhibitor of metalloproteinases linked to the last exon of Thy-1, which encodes for a glycosyl-phosphatidyl-inositol membrane anchor. Infection of postmitotic neocortical and hippocampal neurons in low-density primary cultures with these vectors, achieved reliable expression of all three foreign gene products in various neocortical cell types, e.g. pyramidal neurons, non-pyramidal neurons, and glial cells. The percentage of neurons expressing transgenes ranged from 1 to 46% depending on the multiplicity of infection (highest assayed = 5); the percentage of glial cells expressing transgenes ranged from 0.5 to 98% (highest multiplicity assayed = 3.4). Expression of transgenes could be detected for up to three days in approximately 20% of neurons infected at a multiplicity of infection of 1. Infection of neurons with tk K-derived recombinant vectors inhibited their protein synthesis by 40-50% at a multiplicity of infection of 10, but no effect was observed at a multiplicity of infection of 1. Infection of glial cells with the same vectors at a multiplicity of infection of 1 inhibited protein synthesis by more than 90%. Analysis of neuronal viability at different times post-infection indicated that more than 98% of neurons expressing transgenes 48 h post-infection were viable. Thus, low-density neuronal cultures can be used to assess the efficiency of herpes simplex virus type 1-derived gene transfer vectors and transgene expression in developing cortical postmitotic cells, before and after they establish polarity. In addition, we show that two cytoplasmic enzymes, beta-galactosidase and chloramphenicol acetyl transferase, are able to diffuse freely in the cytoplasm reaching even growth cones in young neurons, while the chimeric protein tissue inhibitor of metalloproteinases/Thy-1 is correctly targeted to the plasma membrane via a glycosyl-phosphatidylinositol anchor. This model system should be useful for investigation of cellular and molecular aspects of the development and establishment of neuronal polarity, as well as for analysis of signals involved in protein targeting in postmitotic neurons.

Identification of two protein binding sites within the varicella-zoster virus major immediate early gene promoter.

Binding sites for cellular proteins in the promoter of the varicella-zoster virus (VZV) major immediate early (IE) gene were investigated. Protein binding was detected at sequence motifs possessing homology to the CCAAT element and an ATF/AP-1-like binding site, and recognition of the ATF/AP-1 site was apparently facilitated by occupation of the CCAAT site. Gene expression directed by the VZV major IE promoter was stimulated by the adenovirus 5, 289 amino acid EIA gene product. The implications of the results for VZV gene expression and replication are discussed.

A non-cytotoxic herpes simplex virus vector which expresses Cre recombinase directs efficient site specific recombination.

The coding sequences for the bacteriophage P1 recombinase Cre were cloned into the genome of a herpes simplex virus type 1 (HSV-1) mutant which is severely impaired for the synthesis of immediate early (IE) proteins. The resulting recombinant, virus in1372, expressed functional Cre which mediated the excision in trans of loxP-flanked sequences located in the HSV-1 genome, both in tissue culture cells and in vivo in mouse sensory neurons. Infection with in1372 also resulted in recombination, at high efficiency, between loxP sequences in the cellular genome without causing detectable cytotoxicity. Mutant in1372 is a versatile vector for the delivery of Cre in tissue culture and in vivo.

A general survey of the proton spin-lattice relaxation-times of some oligo- and poly-saccharide derivatives.

A Fourier-transform method has been used to measure the spin-lattice relaxation-times (T1 values) of the anomeric protons of a selection of oligo- and poly-saccharide derivatives. Although systematic variations are found for the substances of lower molecular weight, these variations are essentially non-existent at higher molecular weights. Data for the disaccharides cellobiose, maltose, lactose, gentiobiose, and melibiose demonstrate that proton T1-values may provide a powerful method for evaluating conformations of oligosaccharides.

Chemical and carbon-13 cross-polarization magic-angle spinning nuclear magnetic resonance characterization of logyard fines from British Columbia.

Phasing out beehive burners and rising costs for landfilling have increased the need to widen options for utilization of the smaller size fractions of woody wastes generated during log handling and sawmilling in British Columbia. We characterized several size classes of logyard fines up to 16 mm sampled from coastal and interior operations. Total C, total N, ash, and condensed tannin concentrations were consistent with properties derived largely from wood, with varying proportions of bark and mixing with mineral soil. Especially for < 3-mm fractions, the latter resulted in high ash contents that would make them unsuitable for fuel. Solid-state 13C cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectra were consistent with the chemical data, with high O-alkyl intensity and similarity to naturally occurring woody forest floor; no samples were high in aromatic or phenolic C. Aqueous extracts of two < 16-mm fines, which accounted for only a small proportion of the total C, were enriched in alkyl C and had low or undetectable tannins. Application to forest sites might cause short-term immobilization of N, but also might include possible longer-term benefits from reduction of N loss after harvesting and restoration of soil organic matter in degraded sites.

Decomposition and nitrogen mineralization from biosolids and other organic materials: relationship with initial chemistry.

Biosolids are effective forest fertilizers. In order to facilitate their use it is important that one be able to predict the amount and rate of mineralization of nutrients, particularly nitrogen, and the relationship between substrate chemistry and N release. We examined the relationships between substrate quality and nitrogen release in a variety of organic materials. Rates of decomposition and net N mineralization from four biosolids, wheat straw, paper fines, and Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] needle litter were measured during 391-d incubations in a greenhouse, and at two field sites in wet coastal and dry interior forests. Decomposition rates were best predicted by a model incorporating the ratio of carbon to organic matter. The decomposition model extrapolated well to the field when site-specific correction factors were applied. There was a weak relationship between rates of decomposition and net N mineralization. Rates of net N mineralization were best predicted by a model incorporating the initial organic N concentration and the proportion of phenolic C determined from solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. The mineralization model extrapolated less well to the field, but the effect of substrate chemistry was still apparent. Among the four biosolids there was a strong correlation between organic N concentration and indices or protein determined from 13C NMR, suggesting that these protein indices may be useful for predicting N mineralization from biosolids. There was some evidence that the protein content and N mineralization in biosolids may be predictable from the sewage treatment process employed.

Harvesting and climate effects on organic matter characteristics in British Columbia coastal forests.

As part of investigations into the effects of harvesting old-growth forest, we characterized carbon in five organic matter pools in eight forest chronosequences of coastal British Columbia. Each chronosequence comprised stands in four seral stages from regeneration (3-8 yr) to old-growth (>250 yr), with second-growth stands mostly of harvest origin. Stands were located in two biogeoclimatic subzones with contrasting climate (wetter, slightly cooler conditions on the west coast of Vancouver Island than on the east). Carbon concentrations in fine woody debris (FWD), forest floor (LFH), fine roots from LFH, and two water-floatable fractions from 10 to 30 cm mineral soil (MIN-ROOT, 2-8 mm and MIN-FLOAT, <2 mm) showed no significant effects due to climate, seral stage, or site. There were some significant differences in N concentrations, but none related to seral stage. Carbon-13 cross-polarization with magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectra with principal component analysis of relative areas also showed little harvesting effect, but greater variation related to input of coarse woody debris (CWD) vs. roots high in tannin. Overall, there tended to be more spectral features associated with wood and lignin in the west; whereas some MIN-ROOT samples from the drier east side had aromatic intensity attributed to charcoal. The minimal effects of one harvest on organic matter are most likely due to the large legacy effect; however, more intensive management will probably result in less CWD retention, less charcoal input, and less microsite variability in these pools of poorly decomposed organic matter.

Soil enzyme inhibition by condensed litter tannins may drive ecosystem structure and processes: the case of Kalmia angustifolia.

Kalmia angustifolia is an ericaceous shrub that can rapidly spread on recently harvested boreal forest sites, causing a slow-down in soil nutrient cycling and reduced growth of spruce seedlings. It has been hypothesized that tannins released from Kalmia litter suppress soil enzyme activity, and are thus important in controlling ecosystem structure and processes. Here the effects of different concentrations of tannins extracted from both Kalmia and black spruce (Picea mariana) foliage were tested on enzyme activities of soil extracts. Then the effects of various Kalmia-black spruce litter mixtures on soil enzyme activity were investigated. Lastly, the correlation between Kalmia cover in the field and soil enzyme activity was measured. Both tannin types suppressed beta-glucosidase and acid phosphatase activities, and the magnitude of these effects was concentration-dependent. beta-glucosidase and amidase activity decreased linearly with an increasing Kalmia : spruce litter ratio added to soil. A field survey of 24 sites revealed a negative relationship between percentage Kalmia cover and beta-glucosidase activity. Collectively, results of the three experiments converge to support the claim that enzyme inhibition by litter tannins has evolved as an important mechanism controlling ecosystem processes and structure following Kalmia invasion on recently disturbed forest sites.

Control of herpes simplex virus type 1 mRNA synthesis in cells infected with wild-type virus or the temperature-sensitive mutant tsK.

This paper deals with control of mRNA levels, assayed by in vitro translation, in cells infected with herpes simplex virus type 1 (HSV-1). A particularly useful marker has been pyrimidine deoxyribonucleoside kinase (dPyK) mRNA, for which the enzymatically active product can be assayed quantitatively. Cells infected with the HSV-1 temperature-sensitive mutant tsK at the nonpermissive temperature (38.5 degrees C) or with wild-type HSV-1 in the continuous presence of cycloheximide contained no detectable dPyK mRNA. Upon temperature shift-down of tsK-infected cells to 31 degrees C, dPyK mRNA was produced, and this event was inhibited by actinomycin D but not cycloheximide. This result demonstrated that the defective polypeptide in tsK-infected cells was involved in transcription of the dPyK gene and could regain activity at 31 degrees C. Because tsK-infected cells synthesized mainly immediate early polypeptides at 38.5 degrees C, the involvement of this polypeptide class in synthesis of dPyK mRNA was investigated. Analysis of the kinetics of inductions of dPyK mRNA indicated that the temperature-sensitive lesion in tsK lies in an immediate early polypeptide which is directly responsible for activation of the dPyK gene at the transcriptional level.

Abnormal properties of an immediate early polypeptide in cells infected with the herpes simplex virus type 1 mutant tsK.

Previous studies (R. J. Watson and J. B. Clements, Virology 91:364--379, 1978; C. M. Preston, J. Virol. 29:275--284, 1979) have shown that the herpes simplex virus type 1 (HSV-1) mutant tsK has a temperature-sensitive lesion in an immediate early polypeptide whose function is to induce synthesis of new viral transcripts, including mRNA, for pyrimidine deoxyribonucleoside kinase. The studies presented here examine the properties of immediate early polypeptides in wild-type HSV-1 and tsK-infected cells at 31 and 38.5 degrees C. The overall pattern of immediate early protein synthesis was similar in wild-type HSV-1- and tsK-infected cells when radiolabeled with [35S]methionine or 14C-amino acid mixture. Further investigation, however, revealed two aberrant properties of the polypeptide Vmw 175 in tsK-infected cells at 38.5 degrees C. Upon cell fractionation, large amounts of this polypeptide were recovered in the cytoplasmic fraction, in contrast to tsK-infected cells at 31 degrees C or wild-type HSV-1-infected cells at either temperature. Furthermore, at 38.5 degrees C tsK-induced Vmw 175 was not processed normally to forms of lower electrophoretic mobility. Both of these defects were reversible upon downshift of tsK-infected cells, even in the absence of further protein synthesis, but were not observed in cells infected with a revertant of tsK. Coinfection of tsK-infected cells with wild-type HSV-1 did not alleviate these lesions, suggesting that they resulted from an abnormal Vmw 175 polypeptide rather than from a defective processing enzyme. Temperature upshift of tsK-infected cells caused reversion of Vmw 175 to the mutant form. The progression to synthesis of late polypeptides was also arrested; therefore, a functional lesion was also reversible upon temperature changes between 31 and 38.5 degrees C during the early stages of infection. The identification of a polypeptide with abnormal properties in tsK-infected cells and the demonstration that these properties, and the functional lesion, are reversible may provide an important system for investigation of HSV-1 transcriptional control.

Cell-free synthesis of herpes simplex virus-coded pyrimidine deoxyribonucleoside kinase enzyme.

The incubation of a cell-free protein-synthesizing system prepared from rabbit reticulocytes with cytoplasmic RNA from herpes simplex virus (HSV)-infected cells resulted in increased thymidine kinase activity. This enzyme activity was specifically inhibited by anti-HSV antiserum and was relatively unaffected by TTP, an inhibitor of cellular thymidine kinases. Induction of the new activity was prevented by addition of inhibitors of eucaryotic protein synthesis, and no new activity was detected when RNA from cells infected with pyrimidine deoxyribonucleoside kinase-deficient mutants, instead of wild-type HSV, was added. An increased deoxycytidine kinase activity with similar properties to the HSV-specified enzyme activity was also present in cell-free systems incubated with RNA from HSV-infected cells. Phosphorylation of thymidine and deoxycytidine at 30 degrees C continued for longer than 11 h. The findings are consistent with the accurate synthesis in vitro of enzymically active HSV-specified pyrimidine deoxyribonucleoside kinase.

Cell-free systems for in vitro translation of herpes simplex virus messenger RNA.

A cell-free system active in translation of HSV mRNA was obtained by fractionation of reticulocyte lysates. Endogenous protein synthesis was further reduced by preincubation with micrococcal nuclease, although this treatment did not affect the translation of infected (or uninfected) BHK cell RNA. Polypeptides synthesized by the fractionated reticulocyte cell-free system were very similar to those produced by unfractionated reticulocyte lysates.

Retention of nonlinear viral DNA during herpes simplex virus latency in vitro.

We investigated the state of the herpes simplex virus type 2 (HSV-2) genome during latency in vitro. Latent DNA was present in the cell nucleus in a nonlinear configuration. The joint fragment was represented at approximately double the molar concentration of fragments from unique regions, indicating that genome termini had fused. The HSV-2 genome copy number was estimated to be between 1.5 and 8, with a mean value of 4.5 per latently infected cell. Nonlinear HSV DNA can therefore exist during latency both in vivo and in vitro.

Establishment of latency in vitro by the herpes simplex virus type 1 mutant in1814.

The herpes simplex virus type 1 (HSV-1) mutant in1814 possesses an insertion mutation that abolishes trans-activation of immediate early (IE) transcription by the virion protein Vmw65. Interactions between in1814 and the host cell were examined by use of an in vitro latency system which relies on infection of human foetal lung (HFL) cells at 42 degrees C to prevent lytic growth of virus. Mutant in1814 was retained in HFL cells after infection at low m.o.i. and incubation at 42 degrees C, and was reactivated by superinfection of monolayers with viruses that express the HSV-1 IE protein Vmw110. Moreover, latency was established by in1814 in an analogous manner at 37 degrees C. The low cytotoxicity of in1814 enabled an investigation of latency after infection at high m.o.i. (five particles per cell) to be undertaken. At 42 degrees C, or at 37 degrees C in the presence of an inhibitor of DNA synthesis, in1814 DNA was maintained at low abundance (one to eight copies per infected cell) in a non-linear configuration. The absence of trans-activation by Vmw65 therefore predisposes HSV to latency, as opposed to lytic growth, in HFL cells, resulting in the retention of the genome in a form resembling that found in vivo.